Although necrosis and necroinflammation are central features of many liver diseases, the role of programmed necrosis in the context of inflammation-dependent hepatocellular death remains to be fully determined. Here, we have demonstrated that the pseudokinase mixed lineage kinase domain–like protein (MLKL), which plays a key role in the execution of receptor-interacting protein (RIP) kinase–dependent necroptosis, is upregulated and activated in human autoimmune hepatitis and in a murine model of inflammation-dependent hepatitis. Using genetic and pharmacologic approaches, we determined that hepatocellular necrosis in experimental hepatitis is driven by an MLKL-dependent pathway that occurs independently of RIPK3. Moreover, we have provided evidence that the cytotoxic activity of the proinflammatory cytokine IFN-γ in hepatic inflammation is strongly connected to induction of MLKL expression via activation of the transcription factor STAT1. In summary, our results reveal a pathway for MLKL-dependent programmed necrosis that is executed in the absence of RIPK3 and potentially drives the pathogenesis of severe liver diseases.

The polarization of macrophages is regulated by transcription factors, such as NF-κB and activator protein 1 (AP-1). In this manuscript, we delineated the role of the transcription factor Fos-related antigen 1 (Fra-1) during macrophage activation and development of arthritis. Network level interaction analysis of microarray data derived from Fra-1– or Fra-2–deficient macrophages revealed a central role of Fra-1, but not of Fra-2, in orchestrating the expression of genes related to wound response, Toll-like receptor activation, and interleukin signaling. ChIP sequencing and standard ChIP analyses of macrophages identified arginase 1 (Arg1) as a target of Fra-1. Luciferase reporter assays revealed that Fra-1 downregulated Arg1 expression by direct binding to the promoter region. Using macrophage-specific Fra-1– or Fra-2–deficient mice, we observed enhanced expression and activity of Arg1 and a reduction of arthritis in the absence of Fra-1, but not of Fra-2. This phenotype was reversed by treatment with the arginase inhibitor N^ω-hydroxy-nor-ʟ-arginine, while ʟ-arginine supplementation increased arginase activity and alleviated arthritis, supporting the notion that reduced arthritis in macrophage-specific Fra-1–deficient mice resulted from enhanced Arg1 expression and activity. Moreover, patients with active rheumatoid arthritis (RA) showed increased Fra-1 expression in the peripheral blood and elevated Fra-1 protein in synovial macrophages compared with RA patients in remission. In addition, the Fra-1/ARG1 ratio in synovial macrophages was related to RA disease activity. In conclusion, these data suggest that Fra-1 orchestrates the inflammatory state of macrophages by inhibition of Arg1 expression and thereby impedes the resolution of inflammation.

Arginase 1 (Arg1), which converts l-arginine into ornithine and urea, exerts pleiotropic immunoregulatory effects. However, the function of Arg1 in inflammatory bowel disease (IBD) remains poorly characterized. Here, we found that Arg1 expression correlated with the degree of inflammation in intestinal tissues from IBD patients. In mice, Arg1 was upregulated in an IL-4/IL-13^– and intestinal microbiota–dependent manner. Tie2-Cre Arg1^fl/fl mice lacking Arg1 in hematopoietic and endothelial cells recovered faster from colitis than Arg1-expressing (Arg1^fl/fl) littermates. This correlated with decreased vessel density, compositional changes in intestinal microbiota, diminished infiltration by myeloid cells, and an accumulation of intraluminal polyamines that promote epithelial healing. The proresolving effect of Arg1 deletion was reduced by an l-arginine–free diet, but rescued by simultaneous deletion of other l-arginine–metabolizing enzymes, such as Arg2 or Nos2, demonstrating that protection from colitis requires l-arginine. Fecal microbiota transfers from Tie2-Cre Arg1^fl/fl mice into WT recipients ameliorated intestinal inflammation, while transfers from WT littermates into Arg1-deficient mice prevented an advanced recovery from colitis. Thus, an increased availability of l-arginine as well as altered intestinal microbiota and metabolic products accounts for the accelerated resolution from colitis in the absence of Arg1. Consequently, l-arginine metabolism may serve as a target for clinical intervention in IBD patients.

Activation of NK cells is a hallmark of infections with intracellular pathogens. We previously showed that the protozoan parasite Leishmania infantum triggered a rapid NK‐cell response in mice that required TLR9‐positive myeloid DC and IL‐12, but no IFN‐α/β. Here, we investigated whether IL‐15 or IL‐18 mediate the activity of IL‐12 or function as independent activators of NK cells. In contrast to earlier studies that described IL‐15 as crucial for NK‐cell priming in response to TLR ligands, the expression of IFN‐γ, FasL, perforin and granzyme B by NK cells in L. infantum‐infected mice was completely preserved in the absence of IL‐15, whereas the proliferative capacity of NK cells was lower than in WT mice. IFN‐γ secretion, cytotoxicity and FasL expression of NK cells from infected IL‐18^−/− mice were significantly reduced compared with controls, but, unlike IL‐12, IL‐18 was not essential for NK‐cell effector functions. Part of the NK‐cell‐stimulatory effect of IL‐12 was dependent on IL‐18. We conclude that IL‐15 is not functioning as a universal NK‐cell priming signal and that IL‐18 contributes to the NK‐cell response in visceral leishmaniasis. The cytokine requirements for NK‐cell activation appear to differ contingent upon the infectious pathogen.

Natural killer (NK) cells are important components of a protective immune response against intracellular pathogens such asLeishmaniaparasites, which reside within myeloid cells. Previousin vivostudies in murine cutaneous or visceral leishmaniasis showed that NK cells are activated by conventional dendritic cells in a Toll-like receptor 9-, interleukin-12 (IL-12)-, and IL-18-dependent manner during the early phase of infection and help to restrict the tissue parasite burden by unknown mechanisms. Here, we tested whether NK cells contribute to the control ofLeishmaniainfections by lysing or by activating infected host cells. Coculture experiments revealed that activated NK cells from poly(I:C)-treated mice readily killed tumor target cells, whereasLeishmania infantum- orL. major-infected macrophages or dendritic cells remained viable. Infection withLeishmaniadid not significantly alter the expression of NK cell-activating molecules (retinoic acid early transcript alpha [Rae-1α], mouse UL16-binding protein-like transcript 1 [MULT-1], CD48) or inhibitory molecules (major histocompatibility complex [MHC] class I, nonclassical MHC class 1b molecule Qa-1) on the surface of myeloid cells, which offers an explanation for their protection from NK cell cytotoxicity. Consistent with thesein vitrodata,in vivocytotoxicity assays revealed poor cytolytic activity of NK cells against adoptively transferred infected wild-type macrophages, whereas MHC class I-deficient macrophages were efficiently eliminated. NK cells activated by IL-12 and IL-18 stimulated macrophages to kill intracellularLeishmaniain a cell contact-independent but gamma interferon-, tumor necrosis factor-, and inducible nitric oxide synthase-dependent manner. We conclude thatLeishmaniaparasites, unlike viruses, do not render infected myeloid cells susceptible to the cytotoxicity of NK cells. Instead, soluble products of NK cells trigger the leishmanicidal activity of macrophages.

Tuberculosis patients and mice infected with live Mycobacterium tuberculosis accumulate high numbers of myeloid-derived suppressor cells (MDSCs). Here, we hypothesized that dead M . tuberculosis vaccines also may induce MDSCs that could impair the efficacy of vaccination. We found that repeated injections of M . tuberculosis vaccines (heat-killed M . tuberculosis in incomplete Freund’s adjuvant, such as Montanide) but not single or control vaccines without M . tuberculosis strongly expanded CD11b ⁺ myeloid cells in the spleen, leading to T cell suppression of proliferation and killing ex vivo. Dead M . tuberculosis vaccination induced the generation of CD11b ⁺ Ly6C ^hi CD115 ⁺ iNOS/ Nos2 ⁺ monocytic MDSCs (M-MDSCs) upon application of inflammatory or microbial activation signals. In vivo these M-MDSCs were positioned strategically in the splenic bridging channels and then positioned in the white pulp areas. Notably, within 6–24 hours, in a Nos2 -dependent fashion, they produced NO to rapidly kill conventional and plasmacytoid DCs while, surprisingly, sparing T cells in vivo. Thus, we demonstrate that M . tuberculosis vaccine induced M-MDSCs do not directly suppress effector T cells in vivo but, instead, indirectly by killing DCs. Collectively, we demonstrate that M . tuberculosis booster vaccines induce M-MDSCs in the spleen that can be activated to kill DCs. Our data suggest that formation of MDSCs by M. tuberculosis vaccines should be investigated also in clinical trials.